Purification and mass spectrometric analysis of the μ opioid receptor

Keith H. Christoffers, Hong Li, Susan M. Keenan, Richard D. Howells

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

A mouse μ opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B max of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged μ opioid receptor with a K D of 1.0 nM. The agonists [D-Ala2,N-Me-Phe 4,Gly5-ol]enkephalin (DAMGO), morphine and [D-Ala 2,D-Leu5]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged μ receptor with KI's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged μ receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The μ receptor was solubilized from HEK 293 cell membranes with n-dodecyl-β-D-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)-agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified μ opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the μ opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the μ opioid receptor.

Original languageEnglish (US)
Pages (from-to)119-131
Number of pages13
JournalMolecular Brain Research
Volume118
Issue number1-2
DOIs
StatePublished - Oct 21 2003

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Keywords

  • FLAG epitope
  • Human embryonic kidney
  • Nickel-nitrilotriacetic acid

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